SNPs detection by a single-strand specific nuclease on a PNA zip-code microarray.
In this report, a reliable peptide nucleic acid (PNA) microarray-based method for accurately detecting single nucleotide polymorphism (SNP) in human genes is described. The technique relies on the mismatched cleavage activity of a single-strand specific (SSS) nuclease. PCR amplification was performed to prepare gene fragments containing the mutation sites. The amplified fragments were then employed as templates for the SSS nuclease reaction using chimeric probes, modified with biotin at the 5' end and extended with a unique anchoring zip-code complement sequence at the 3' end. The SSS nuclease promotes cleavage of heteroduplex DNAs at base mismatched positions to produce crumbled chimeric probes in the presence of imperfectly matching template strands. In contrast, the probes remain intact when they interact with perfectly matched template strands. Only the non-fragmented probes generate fluorescence signals after treatment with streptavidin-Cy3 on the PNA zip-code array. This methodology was used to successfully genotype selected Korean-specific BRCA mutation sites with wild type and mutant samples. The investigation has led to the development of a reliable SSS nuclease-based system for the diagnosis of human genetic mutations or SNPs.
Mun
Hyo Young
HY
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Yuseong-gu, Daejeon, Republic of Korea.
Girigoswami
Agnishwar
A
Jung
Cheulhee
C
Cho
Dae-Yeon
DY
Park
Hyun Gyu
HG
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000295:instrumentation*; D003851:Deoxyribonucleases / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D020135:Peptide Nucleic Acids; D004247:DNA; D003851:Deoxyribonucleases